Cardiovascular instability is one of the most difficult challenges in clinical management of pre-term infants due to unique neonatal patterns of cardiovascular reactivity, particularly in the cerebral circulation. Compared to adult arteries, immature cerebral arteries exhibit greater reliance on Ca++ influx than on intracellular Ca++ release for initiation of contraction, but also exhibit markedly enhanced myofilament Ca++ sensitivity. One possible explanation for this enhanced fetal myofilament Ca++ sensitivity is that phosphorylation of regulatory myosin light chain (MLC20), a key contractile protein, is upregulated in immature cerebral arteries. Previous attempts to study phosphorylation of MLC20 in situ have been limited by technical barriers, including the inability to measure the rapid time course of MLC20 phosphorylation in intact arteries. Methods recently developed in the applicant's laboratory have surmounted these problems. A custom designed apparatus now can simultaneously measure cytosolic Ca++ transients and MLC20 phosphorylation with millisecond resolution in intact arteries in a cuvette small enough to accommodate fetal cerebral arteries. Protein expression systems are now available that can produce single isoforms of MLC20 in quantities suitable for kinetic measurements. New confocal colocalization methods are capable of resolving interactions among multiple different contractile proteins in these small arteries. Based on these exciting new methods, this proposal was designed to explore the main hypothesis that myofilament Ca++ sensitivity is upregulated in immature compared to mature cerebral arteries due to an increased velocity of MLC20 phosphorylation. A corollary hypothesis supported by strong preliminary data predicts that the ultra-structural organization of MLC20 within cerebrovascular smooth muscle is an important determinant of its ability to serve as a substrate for phosphorylation. To address these ideas, three specific aims have been designed: 1) determine substrate- velocity relations for MLC20 phosphorylation in broken cell preparations in the presence and absence of phosphatase inhibitors with age-specific sources of purified ovine MLC20 and synthesized single isoforms of Non-Muscle and Smooth-Muscle MLC20; 2) determine the velocities of MLC20 phosphorylation in intact cell preparations in the presence and absence of phosphatase inhibitors, in response to both spontaneous and standardized cytosolic Ca++ transients; and 3) determine the abundance, distribution, and co-localization of MLC20 in relation to myosin light chain kinase, myosin light chain phosphatase, smooth muscle 1-actin, and myosin heavy chain isoforms. These experiments will be conducted in middle cerebral arteries from term fetal lambs, newborn lambs, and non-pregnant adult sheep to define the effects of postnatal age on these regulatory mechanisms. Further understanding of these mechanisms offers potential to refine clinical strategies for management of infants at risk for cerebrovascular injury, as well as those afflicted by numerous other diseases such as asthma, sepsis, and colitis, in which altered rates of MLC20 phosphorylation are a key factor.